Lipid peroxidation contributes to the evolution of secondary damage in traumatic brain injury (TBI) however; essential information on molecular targets of oxidation is largely unknown. We reported that two anionic phospholipids-mitochondrial cardiolipin (CL) and extramitochondrial phosphatidylserine (PS) - are major targets of TBI-induced oxidation in brain. These oxidation reactions, catalyzed by complexes of cytochrome c (cyt c) with CL and PS, are associated with mitochondrial stages of programmed cell death and recognition of damaged cells by professional phagocytes, respectively. Studies in experimental TBI have revealed that the cortex, hippocampus and thalamus are selectively vulnerable to injury. However, information on spatial distribution of phospholipids and their oxidation products in various brain regions is lacking. The goal of this application is to fill thi gap of knowledge by developing and applying a new technology - imaging mass spectrometry (IMS) - for spatial and temporal mapping of diverse molecular species of phospholipids and their oxidation products and superimposing them onto neuropathology of the injured brain. This information will be critical for the design and development of targeted antioxidant therapies and evaluating their efficacy in TBI. We will use the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS (Bruker Solarix) for a panoramic snap-shot of thousands of lipid signals simultaneously to obtain lipid maps of the brain. We will complement these studies by novel IMS technologies with improved spatial resolution: i) Matrix Assisted Laser Desorption Ionization-Postionization-Ion Mobility- orthogonal Time of Flight MS (MALDI-POST-IM-oTOFMS) with employment of nano-scale matrices; ii) oversampling-laser stepping MALDI-FTICR, and iii) micro-deposition of matrix. We will merge this information with fluorescent microscopic imaging to reveal structure and metabolic function of the vulnerable brain regions. This will be the first comprehensive lipidomics, oxidative lipidomics and IMS analysis of CL and PS in different brain regions. This enabling technology will resolve issues of spatial confinements of peroxidation reactions in lipids in the brain that cannot otherwise be readily examined. We will also examine brain tissue removed from TBI patients with refractory intracranial hypertension and brain-bank control tissue using oxidative lipidomics and IMS. As lipids and oxidized lipids are vital signaling molecules, the development of such technology and new information on the biochemistry of lipids should be of broad fundamental interest. Our progress with novel mitochondria targeted electron scavengers (gramicidin conjugated nitroxides) and inhibitors of cyt c/ CL peroxidase (triphenylphosphonium conjugated imidazole fatty acids) will facilitate our ability to pharmacologically delineate the roe of intracellular oxidized CL and PS in TBI. IMS used in a metabolomic mode towards these small molecules will reveal critical molecular pharmacologic information on the disposition and efficacy of these putative neuroprotectants against TBI. Overall, IMS technology and the underlying contribution of dyshomeostasis of mitochondrial CL and extramitochondrial PS are likely to be important for TBI studies and have implications for other CNS disorders. PUBLIC HEALTH RELEVANCE: Severe traumatic brain injury (TBI) is the leading cause of death in children and young adults. Lipid peroxidation contributes to evolution of secondary damage after TBI. The goal of this application is to develop and apply a new technology - imaging mass spectrometry (IMS) - for spatial and temporal mapping of diverse molecular species of phospholipids and their oxidation products and superimposing them onto neuropathology of the injured brain. This enabling technology will resolve issues of spatial confinements of peroxidation reactions in lipids in the brain that cannot otherwise be readily examined. This information is critical for the design and development of targeted antioxidant therapies and evaluating their efficacy in TBI. Novel mitochondria targeted therapies will be tested for their neuroprotective efficacy using these approaches.